Greetings,
I am attempting to balance the relative advantages (and cost!) of the systems for ISH in whole tissue (early embryos). Most of our probes would end up being custom given that we work in an alternative model organism. In general it would be nice to have a reliable single channel as well as a system that could be multiplexed to compare spatial expression within the same tissue/organism? Any thoughts?
Hi Tim,
I have experience with HCR and TSA. Both work fine with their own pros and cons but mainly dependent upon the expression of gene and tissue type. I would suggest starting with HCR. It would cheap in long run, easy to customize Oligo probes and simple and steps to run the experiment specially for thin sections or simpler tissues. You can custom design your oligos and get it synthesized from IDT and get the DNA hairpins from Molecular Instruments (start with AlexaFluor 647 to test). May be order the Oligo probes for first time from them too if you are not comfortable designing your own, with the later option you will have a ton of probes to test and work.
TSA system is robust but optimizing the HRP step will require some work as there is always high chances of getting non specific precipitation, but again it depends upon the gene.
RNAScope is well validated from the vendor itself, if you have funding and urgent need to run the experiments then you can try that too as first step.
For more detailed insight on HCR, you can look at my paper, links below-
Article Optimization and evaluation of fluorescence in situ hybridiz...
Article Optimization and evaluation of fluorescence in situ hybridiz...
Good Luck!
Dear Tim,
we are specialized in designing smFISH probes for custom gene, HuluFISH. it works up to 7 gene detection in one reaction on 3 channel microscope. if you would have a microscope with more channels, you can even detect more genes at once.
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